Composition usable in the treatment of cellular degeneration

ABSTRACT

The invention relates to a composition for use in the very rapid treatment of cell degeneration, in particular in oncologic field, characterized in that it is constituted by a compound of Potassium Bicarbonate and D-Ribose.

The present invention relates to the use of a composition as a drug forrapidly combating the tissue degeneration; in particular, the drug isparticularly useful in cases of degenerative diseases caused byparticular conditions of stress, such as, for example, the exposure toionizing radiations (gamma rays and X-rays), exposure to UV, or cellulardamage caused by toxic molecules, etc.

It is known that radiations in general, as well as other endogenous andexogenous factors, produce damages at the cellular level forming freeradicals, highly reactive molecules which produce chemical oxidations oflipids and proteins, resulting in oxidative stress. The general effectsthus produced in the organism are mainly the following: a weakening ofthe immune system; a stepped-up physiological organic deterioration; theproduction of degenerative forms.

A particularly significant example relates to the cancer induction bythe radiations, mainly the ionizing radiations (X and γ rays for theelectromagnetic radiation, α e β rays for the material particles), aswas indeed highlighted by the researchers who first worked withradio-emitting materials and as has been documented by a huge amount ofscientific works of the last seventy years.

A dramatic and particular example of this causal relationship betweenradiation exposure and increased incidence of cancer is related to theeffects of radiation on healthy cells. It is known that the radiation isintended to stop the growth of a tumor, but since it is not possible todiscriminate with absolute precision, in the treatment plan, the cellsto be treated from the nearby healthy ones, it is necessary that theabsorbed dose the system does not exceed a given value (usuallyindicated in 2 Gy, corresponding to 200 Rad) to prevent the irradiationof healthy tissue causes a rapid new carcinogenesis. In oncologicradiation it is known the possibility that a new cancer can develop intissues that were directly exposed to the treatment (radiogenicpathogenesis), but it is also possible that an indirect process causes asecond tumor in a different place of occurrence. It is thereforeimportant and essential to prevent and reduce the effects of theexposure to healthy tissue during radiation therapy.

Among the effects of exposure to radiation or, in general, to oxidativestress, are to be considered the bio-physical and chemical changes thatcan result in damage to cell membranes and to the DNA. In fact, in thepresence of an oxidizing stress, the NA+-K+ pumps do not perform itstask correctly and there occurs an imbalance of the four fundamentalcations for a well-balanced cellular functioning, namely Sodium,Potassium,

Calcium and Magnesium. Especially in presence of degenerative diseasesand in particular neoplastic forms, Sodium NA+(a prevalent cation inextracellular fluids) tends to substitute Potassium K+ inside the cell,with a resulting serious cytoplasmic imbalance, which can conduct to asignificant alteration of the intra and extra cellular acid-baseconditions, to a modification of the enzyme and protein function byalteration of the binding sites for the electrolyte and a significantchange of their shape. In addition, a significant decrease in thecytoplasmic concentration of Potassium can cause instability in thedouble helix structure of DNA and, especially, in the action of thetelomerase enzyme, because the Potassium is strongly implicated in thecorrect structure of the G-Quadruplex (sequence of Guanine-rich nucleicacids and stabilized by K+cation), thus prompting mutagenic phenomena.It must also be taken into particular consideration the existence of aSodium-Glucose symport (known in the literature since the 60s of lastcentury) and recently further confirmed by MRI based on sodium (Na²³)and performed on cancer “in vivo” compared with PET on the same tumors,completely overlapping. Aim of the present invention is to eliminate theabove mentioned drawbacks providing with a composition to be used invery rapid treatment of the cellular degeneration, especially inoncologic field, according to the characteristics of claim 1. Theinvention relates to the use of a composition of Potassium Bicarbonateand D-Ribose for forming a drug, in particular an injectable drug, to beused in the very rapid treatment of the cellular degeneration,especially in oncologic field. Other characteristics are described independent claims.

Among the advantages of the present invention, which represents in thissense a novelty in the therapeutic field, there is that the drug carriesout a main action for a rapid decrease not only of the effects caused byfree radicals but, above all, in the development of transformed cells intumoral way. The speed of action of the drug and the subsequent rapiddecrease in tumor cell proliferation was seen “in vitro” on cell lineA72 (canine cancer), with a slowing of proliferation for concentrationsof D-Ribose and KHCO₃ aqueous solution of 0.5 mM and a growth arrest forconcentrations of 5 mM only after 48 hours of treatment. The aspect toemphasize with decision is the novelty of the combination, which is notan obvious extension of a food supplement called Potassium Ascorbatewith Ribose and administered orally, either as antioxidant agent and asa regulator of cellular metabolism. In fact, while until now it wasconsidered that L-Ascorbic acid was the fundamental carrier of Potassiumand that the D-Ribose acted in the compound as a catalyst of theprocess, in this case the total absence of L-Ascorbic acid and the usethe only D-Ribose, to combine with Potassium Bicarbonate (KHCO₃),represents an absolute novelty in therapy. Up to this point has neverbeen taken into account the idea that this monosaccharide could salifyan alkali metal such as Potassium to form the compound PotassiumRibosate. It also presents characteristics of specificity, and also thisis a crucial issue in the therapeutic context, as it acts on cells thathave undergone degenerative changes especially in oncology sense, whileleaving healthy cells unaffected.

This drug binds together the functional characteristics of itscomponents resulting in extremely fast results, not comparable withrespect to the use of components used individually (e.g., an injectabledrug made from potassium chloride and another, even injectable, ribosebased, administered separately, did not produce the same effect); itdoesn't cause toxicity (at prescribed doses) and can be used for longperiods (months and even years) without damage.

Every technician who works in this field will better understand theseadvantages and features and further advantages and features of thepresent invention thanks to the enclosed figures relating to preliminaryresults of the effects of the drug in question on human cells; in thefigures:

FIG. 1 relates to a photo of a control sample;

FIG. 2 relates to a photo of a control sample irradiated with X-ray;

FIG. 3 relates to a photo of a sample treated with the drug of thepresent invention and irradiated with X-ray;

FIG. 4 relates to a photo of a sample treated with the drug of thepresent invention and not irradiated;

FIGS. 5, 6, 7, 8, 9 relate to configurations of D-Ribose.

The following description illustrates the preliminary results of theeffects of D-ribose and KHCO₃ in an aqueous solution on HTB 125irradiated with X-ray.

In particular, the study was directed to the effects of D-Ribose andKHCO₃ in an aqueous solution on normal human breast cells HTB125irradiated with X-ray.

As mentioned above, the radiotherapy can generate secondary tumors(radiogenic pathogenesis) in the same radiation seat or in other seats.It is therefore important and essential prevent and reduce the effectsof exposure to healthy tissue during radiation therapy.

Materials and Methods.

HTB125 cells of mammary gland not tumoral were purchased from AmericanType Culture Collection (ATCC)—Manassas, Va., USA. The cells weremaintained in DMEM (Dulbeccos's Modified Eagle's Medium) purchased fromLonza to which are added 10% fetal calf serum (Lonza), 1% L-glutamine(Sigma Aldrich), 1% Penicillin-Streptomycin (Sigma Aldrich) and 30 μg/mlof epidermal growth factor (Gibco). The cells were incubated at 37° C.in a humidified atmosphere with 5% CO2. The D-ribose was purchased fromSigma-Aldrich and potassium bicarbonate from BDH Prolabo. A stocksolution (K:D-Rib) 250 mM was prepared by dissolving 0.15 mg of D-Riboseand 0.3 mg of KHCO₃, the whole being stirred until the CO₂ is completelyreleased into the air.

The cells were maintained changing the culture media every 48 hours.

The cell population was splitted when it reaches 90-100% of confluence.To split the cell cultures that reached confluence, was uses trypsin(Sigma Aldrich). This enzyme acts as a protease capable of separatingboth the links between cell and substrate as cell-cell.

Treatment and Irradiation of Cell Cultures.

The day before irradiation, cells are seeded 8000/ml and treated with asolution of 5 mM (K: D-Rib). The solution was obtained by diluting thestock 250 mM (K: D-Rib) in DMEM. The day after seeding cells wereirradiated.

In particular, four samples were prepared: control (C, shown in FIG. 1),irradiated control (C_irr, shown in FIG. 2), treated non-irradiated(Krib, shown in FIG. 4) and treated irradiated (Krib_irr, shown in FIG.3). The irradiation was performed with an X-ray tube (Faxitron cabinet43855D) by placing the Petri dish on a rotating plate for ensuringuniformity of dose. The X-ray tube was set to 106 kV and 3 mA. The dosewas equivalent to 1.01 Gy/min and the radiation occurred for a time of 2min. The evaluation of the absorbed dose was carried out via the systemVictoreen NERO mAx Model 8000. In addition, the dose was measured bysimulating the Petri system covering the sensor with the cover of thesame Petri dish. The day after irradiation have changed the means oftreated and untreated samples. The treatment was carried out until theend of the experiment.

Results—Discussion.

To study cell growth and the effect of treatment, the cell growth wascontrolled and in particular the time between irradiation and the firstsplit, i.e. the attainment of confluence. Samples C, Krib and Krib_irrhave reached 90% confluence after 7 days after sowing while the sampleC_irr 10 days after sowing. In addition, the use of trypsin was muchless effective in the samples C, Krib and Krib_irr, compared to thesample C_irr. These results show that the samples C, Krib and Krib_irrhave the same growth kinetics. The kinetics in Krib_irr treated with 5mM (K: D-Rib) was not altered by the radiation; on the contrary thesample C_irr has reached the confluence with a significant delay of 3days. Considering the difference in efficacy of trypsin, it can beassumed a protective effect of the solution (K:D-Rib) on not tumoralcells HTB 125 treated before irradiation and subsequently the same.

The drug of the invention, which in its form of administration isinjectable, consists of a mixture of Potassium Bicarbonate (KHCO₃) andan aldopentose that includes D-ribose (C₅H₁₀O₅). The compound obtainedfrom these two molecules, which is called Potassium Ribosate, is watersoluble and it is administered by injection.

There are no known other trials in which it was administeredintramuscularly or intravenously the composition which constitutes thedrug of the present invention, nor are known other trials that suggestthe use of a drug injection in which the components of PotassiumBicarbonate and D-Ribose provide results so surprising and rapid in theprevention and treatment of degenerative diseases.

It is known that D-Ribose is a pentose sugar which plays a key role inenergy metabolism of the cell (primary component for the production ofATP—adenosine triphosphate) and as a precursor in the biosynthesis ofRNA (ribonucleic acid) and, in the form of deoxyribose, DNA(deoxyribonucleic acid). The D-Ribose is thus simple but it is acompound of fundamental importance in metabolic and functional pathwaysof the cell (especially at the level of production, conversion and useof energy). Precisely in this sense, the mechanisms involved in theKrebs cycle acquire a particular importance. The D-Ribose reveals anactive role in some organic co-factors and its wide distribution in theforms of life on our planet suggests that it may have been involved inprebiotic chemistry on Earth.

The D-Ribose, in addition to the open-chain form (FIG. 5) has other fourisomeric configurations: α and β D-ribopyranose (FIGS. 6 and 7), α and βD-ribofuranose (FIGS. 8 and 9). In living systems ribose is present inthe form β D-ribofuranose, and the open-chain form is able to mediatebetween the other three closed-chain structures for converting suchstructures and promoting the dextrorotatory isomeric β ribofuranosestructure.

D-Ribose and the Potassium Bicarbonate of the present invention are inpure microcrystalline form, readily soluble in water for forminginjectable solutions; the compound is very unstable because of its easyoxidizability and must be stored in suitable protected mono-dosecontainers.

D-Ribose salifies with Potassium Bicarbonate in cold aqueous solutionforming the compound called Potassium Ribosate. Until now this aspect,i.e. the ability of the D Ribose to form a salt, has been completelyoverlooked by the scientific community because it was considered amarginal issue. This effect is made possible thanks to the action of themolecule in the form of open-chain which in aqueous solution, linkingtwo potassium ions, with his rotation to close the ring, gives stabilityand continuity to the β D-ribofuranose form.

Its action has no toxicity (at prescribed doses) and can be used forindeterminate time. It acts rapidly to contrast the uncontrolledproliferation of cells that have undergone neoplastic degeneration,acting on the mechanisms related to the energy metabolism of thesecells, reducing the effect of oxidative stress, enhancing the activityof the immune system and maintaining or restoring the concentration ofintracellular potassium to the correct values.

To better understand these statements it is necessary to remember thatit is known in scientific literature, both in chemical and bio-medicalfield, since the end of 1800 and until the early decades of last century(thanks to the works of Italian Luigi Giacomo Ciamician, born in Triestein 1857 and died in Bologna in 1922) the importance of the pyrrole ringsfor plant and animal life, so that in the 40s of last century NielsTroensegaard developed a hypothesis (improperly called “PyrroleTroensegaard's Hypothesis,” as reported in the appendix by the author of“On the Structure of the Protein Molecule”, Acta Chemica Scandinavica, 1[1947]: 672-682), which is still controversial, hypothesis according towhich it would be precisely the presence of heterocyclic units ofpyrrolic type to constitute the ideal ground for the structure ofproteins. This is also true in the structure of ribonucleic acid (RNA),in which the presence of pyrrolic rings in the helical structure ishighlighted by Purines (Adenine and Guanine).

The heme groups of hemoglobin and chlorophyll contain an iron ion and amagnesium ion bound in a heterocyclic structure known as porphyrin,which consists of four pyrrolic rings linked together and with the ion(iron or magnesium) in the center of the structure. Also some aminoacids (histidine, proline, tryptophan, pyrrolysine) contain a pyrrolicring in their structure. It is also important to note that the blackpigments, skin, hair, moles, etc., are in close relationship with theblacks of Pyrrole and this allows to formulate the hypothesis that thesecompounds are oxidized and poly-condensed pigments with pyrrolicstructure.

It is important to note that the imidic Hydrogen present in thestructure of Pyrrole is easily replaced by the potassium cation(salification process) but not by the sodium cation (Ciamician effect,which occurs in a slightly acidic environment), although these elementsare similar in physico-chemical terms. But, when the local environmentundergoes a change in pH, from neutral or slightly acidic to slightlyalkaline, the sodium can bind to the nitrogen of NH group thustriggering the distortion and the opening of the ring. When the processof carcinogenesis begins, the pyrrolic groups seem to be inactivatedprecisely by an imbalance Sodium/Potassium with changes in acid-baseconditions. Furthermore, the mechanisms that underlie the transformationof the cells with carcinogenic effect find in the polymerization of RNAa key to open the pyrrole rings of Purines.

It is to consider also that Pyrrole, Thiophene and Furan are similar toeach other; in the formation of their compounds, all three follow therule of the similarities of Angeli. It is therefore logical to thinkthat during the process of biologic synthesis of protein derivates ofsuch compounds, similar physico-chemical reactions take place and that,in particular conditions, a pyrrolic group can be replaced by a tiofeneor furanose group.

Potassium Ribosate contains in its molecule a furanose group that can,by analogy, replace one of the pyrrole groups in Potassiun Hemoglobinateor Potassium Proteinate.

Thus Potassium Ribosate, reactivating by analogy the Pyrrole group,restores the phenomena of cell auto-synthesis structuring to physiologicnormality. This is even more important at RNA level because it can limitits uncontrolled polymerization (ifs known that neoplastic cells have agene overexpression and are often multinucleated). Are therefore thesebio-chemical-physical mechanisms, activated by the compound of thepresent invention and which occur in the cell cytoskeleton, to play akey role in inhibiting the uncontrolled proliferation mechanisms; alsothis interpretive key linked to the action of D-Ribose is an absolutenovelty.

Moreover, since the salification is a reversible process, the PotassiumRibosate carried by hemoglobin, by entering the cell restores thebalance between the intermolecular forces of peptide groups presentwithin the cell and restores the concentration of intracellularPotassium to the correct values.

This compound has therefore functions provided with characteristics ofspecificity, since it is able to act on cancer cells to counter theiruncontrolled replication, but at the same acts of normal cells tomaintain their proper electrolyte and electrochemical balance, withoutcreating to the latter any kind of injury in the mechanisms of metabolicregulation, but rather correcting functional imbalances. The inhibitingof the development of degenerative processes show results which arequickly and surprisingly satisfying. It is therefore a very valuable andvery strong cellular antioxidant, which binds together the features ofthe D-Ribose and Potassium, proving much more active of the twoconstituents taken separately. It is evident the synergistic effect ofthe composition of the invention in its use as an injectable drug.

It emerges from stoichiometric calculations that the proportion betweenPotassium Bicarbonate and Ribose must be of a 2:1 ratio, but it ispossible a 3:1 relation.

Regarding the storage and the dosage, the compound of Potassium Ribosatemust be protected from humidity and the sun's rays and dosed ininjectable vials or other suitable separated containers for preparing anextemporary solution of very pure D-Ribose and Bicarbonate of Potassiumaccording to the following preferable proportions. The proportionsprovide to use an injectable vial of Potassium Bicarbonate and D-Ribosecontaining two doses of first component and a dose of the secondcomponent. It is also possible to use three doses of PotassiumBicarbonate and a dose of D-Ribose. Furthermore, it is possible to usean injectable vial of Potassium Bicarbonate containing two doses of thiscomponent and an injectable vial of D-Ribose containing a dose of thesame. It is also possible to use four doses of Potassium Bicarbonate anda dose of D-Ribose. In practice, the details may still vary in anequivalent amount, percentage, type of components used, without goingbeyond the scope of the idea of the solution adopted and thereforeremaining within the limits of the protection afforded by this patent.

1. Composition for use in very rapid treatment of cell degeneration, inparticular in oncologic field, characterized in that it is constitutedby a compound of Potassium Bicarbonate and D-Ribose.
 2. Compositionaccording to claim 1, characterized in that it comprises two parts ofPotassium Bicarbonate and a part of D-Ribose.
 3. Composition accordingto claim 1, characterized in that it comprises three parts of PotassiumBicarbonate and a part of D-Ribose.
 4. Composition according to claim 1,characterized in that it comprises four parts of Potassium Bicarbonateand a part of D-Ribose.
 5. Composition according to one of the precedingclaims, characterized in that it is used to form an intramuscularinjectable drug, which characterizes its rapid responsiveness. 6.Composition according to one of the preceding claims, characterized inthat the components of said compound are in single doses for eachcomponent, associable at the time of use, in order to obtain anextemporaneous solution of a drug.
 7. Use of a composition of PotassiumBicarbonate and D-Ribose in the manufacture of a drug for the very rapidtreatment of cell degeneration, in particular in oncologic field.
 8. Useof a composition of Potassium Bicarbonate and D-Ribose in themanufacture of an injectable drug for the very rapid treatment of celldegeneration, in particular in oncologic field.
 9. Use of a compositionaccording to claim 7 or 8, characterized in that the compositioncomprises two parts of Potassium Bicarbonate and a part of D-Ribose. 10.Use of a composition according to claim 7 or 8, characterized in thatthe composition comprises three parts of Potassium Bicarbonate and apart of D-Ribose.
 11. Use of a composition according to claim 7 or 8,characterized in that the composition comprises four parts of PotassiumBicarbonate and a part of D-Ribose.
 12. Use of a composition accordingto claim 7 or 8, characterized in forming a drug injectable byintramuscular way.
 13. Use of a composition according to claim 7 or 8,characterized in that the components of said compound are in singledoses for each component, associable at the time of use, in order toobtain an extemporaneous solution of a drug.
 14. Injectable drug for usein very rapid treatment of cell degeneration, in particular in oncologicfield, characterized in that it comprises a compound of PotassiumBicarbonate and D-Ribose.